Megakaryocyte (MK) differentiation is a continuous process characterized by sequential steps. MK ploidy increases through endomitosis, with a parallel increase in cell size. Synthesis of storage organelles and plasma membrane is enhanced, resulting in the formation of demarcation membranes. Cytoplasmic maturation is associated with a marked increase in MK size. Finally, mature MK undergo complete cytoskeleton reorganization with microtubule involvement, to induce pseudopodial elongations corresponding to proplatelets (Patel et al., 2005a; Richardson et al., 2005). Platelets are released from the tips of these proplatelets that contain all the platelet organelles.
Circulating platelets are involved in thrombus formation by adhesion to the vessel wall and by aggregation on the first layer of adherent platelets to the subendothelial matrix (Ruggeri, 2003). Von Willebrand factor (VWF) is a major constituent of endothelial cells lining the vasculature. VWF is the only protein able to arrest circulating platelets in conditions of high shear rates (>1000 s−1). Tethered platelets translocate on immobilized matrix VWF, through the binding of GPIb (also known as CD42b) to VWF A1 domain (Huizinga et al., 2002). Activation of the αIIbβ3 integrin (also known as CD41a/CD61), that occurs progressively during transient tethering of platelets, involves GPIb-mediated signalling and allows αIIbβ3 binding to the RGD sequence of VWF. The Bernard-Soulier Syndrome (BSS) is a bleeding disorder characterized by severe thrombocytopenia and giant platelets (Nurden, 2005). It is due to quantitative or qualitative abnormalities of the GPIb-IX-V complex, in particular of the GPIbα subunit that contains the VWF binding site. Normal numbers of MKs are found in the bone marrow of BSS patients, suggesting that the macrothrombocytopenia observed in this setting is related to defective platelet formation from MKs.
Since MK fragmentation into platelets is not often observed in the marrow space, MKs need to migrate into the bone marrow capillaries (Tavassoli and Aoki, 1981). Platelet detachment from proplatelets has been described in the absence of flow conditions. It is intriguing that the yield of platelets shed from cultured MKs in the presence of thrombopoietin (TPO) is far below what could be expected in optimal maturation conditions (Norol et al., 1998). Many steps of platelet shedding remain elusive. In particular a role of shear forces on platelet formation has never been demonstrated. The VWF receptors GPIb and αIIbβ63 are both expressed on the MK surface during maturation (Debili et al., 1990). Few mature MKs have been identified in the blood circulation (Pedersen, 1978; Tavassoli and Aoki, 1981), where they are exposed to VWF on the luminal side of endothelial cells.
Moreover, it is intriguing that in optimal MK culture conditions, the yield of shed platelets in vitro is far below what could be expected from the large daily platelet production in vivo. So, there is an important need of a good system of platelet production.